On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation
- Institute of Molecular Physical Science, Switzerland
- Scientific Center for Optical and Electron Microscopy, Switzerland
- Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, United States
- Institute of Molecular Systems Biology, ETH Zürich, Switzerland
Figures
Figure 1
The polymorphs formed in vitro by wild-type α-synuclein (α-Syn).
The numbered backbone Cα traces depicting a single chain from each of the five types of polymorphs that have been reported to form in vitro with wild-type α-Syn but without cofactors. The interface …
Figure 2
The Type 1 polymorphs.
Backbone Cα traces showing the four different interface polymorphs of Type 1, including 1B whose fold is not classically Type 1 but whose name is kept for consistency with previous publications. The …
Figure 3
3D classification of particles in fibril samples from various conditions.
The 3D classes obtained in RELION for five different fibril samples grown at pH 5.8 and 6.5 in PBS with or without additional NaCl are shown. Each box presents the output classes from one sample …
Figure 4
pH-dependence of observed α-synuclein (α-Syn) polymorphs.
An overview of all of the cryo-electron microscopy (cryo-EM)-resolved polymorphs in this study. The images represent a 9.5 Å slice (about two layers of the amyloid) of the 3D map projected in the Z …
Figure 5
Comparison of the Type 2 and 3 polymorphs.
(A) Protofilaments of polymorphs 2A (blue) and 3B (green) are depicted as Cα traces. The side chains are included on the C-terminal β-strand and the N-terminal segment in the 2A structure in order …
Figure 6
The juvenile-onset synucleinopathy (JOS)-like Type 1M polymorph.
(A) The Type 1M monofilament structure (PDB:8PK2) overlaid with its EM density (including the unmodeled density). The Cα trace is also shown on the left with all K and E side chains indicated as …
Figure 7
The Type 5 polymorph.
The structure of a Type 5 protofilament (PDB:8PK4) overlaid with its EM density. The Cα trace for the two filaments of the Type 5A fibril is also shown to the left. All of the charged (E/D/K) …
Figure 8
Structural variability within the Type 1 polymorphs.
(A) Seven pairwise overlays of the wild-type 1A structure (PDB:6A6B) with other wild-type and mutant structures depicting the range of structural variability that is found in the Type 1 fold. The …
Figure 9
Determination of helical symmetry in Type 3B, 3D, and 5A polymorphs.
2D projections and their Fourier transforms for three different structures (rows) refined with three different types of symmetry (columns). Each of the structures studied was refined as far as …
Figure 10
Cross-seeding does not preserve the seed polymorph.
Fibrils produced at pH 7.4 (F1) and pH 5.8 (F2) were used as seeds to generate new fibril samples with fresh α-synuclein monomer: at pH 7.4 with 5% unfragmented pH 5.8 seeds (F3) or at pH 5.8 with …
Figure 11
2D classes and half-map and model-map FSC curves for dataset 1, Type 3B.
(A) Representative 2D classes of the segments that were used for the 3D reconstruction. (B) The FSC curves produced during postprocessing in RELION with red showing the plot for the phase …
Figure 12
2D classes and half-map and model-map FSC curves for dataset 3, Type 3C.
(A) Representative 2D classes of the segments that were used for the 3D reconstruction. (B) The FSC curves produced during postprocessing in RELION with red showing the plot for the phase …
Figure 13
2D classes and half-map and model-map FSC curves for dataset 5, Type 3D.
(A) Representative 2D classes of the segments that were used for the 3D reconstruction. (B) The FSC curves produced during postprocessing in RELION with red showing the plot for the phase …
Figure 14
2D classes and half-map and model-map FSC curves for dataset 8, Type 5.
(A) The 2D classes of the segments that were used to produce the initial model in relion_helix_inimodel2d. (B) The output of relion_helix_inimodel2d shown as the summed 2D classes and a Z-projection …
Figure 15
2D classes and half-map and model-map FSC curves for dataset 9, Type 1M.
(A) The 2D classes of the segments that were used to produce the initial model in relion_helix_inimodel2d. (B) The output of relion_helix_inimodel2d shown as the summed 2D classes and a Z-projection …
Figure 16
2D classes and half-map FSC curves for dataset 13, Type 2A.
(A) Representative 2D classes of the segments that were used for the 3D reconstruction. (B) The FSC curves produced during postprocessing in RELION with red showing the plot for the phase …
Figure 17
2D classes and half-map FSC curves for dataset 13, Type 2B.
(A) Representative 2D classes of the segments that were used for the 3D reconstruction. (B) The FSC curves produced during postprocessing in RELION with red showing the plot for the phase …
Tables
Table 1
Cryo-electron microscopy (cryo-EM) samples analyzed for this manuscript.
| Dataset | Construct | Batch | Aggregation condition | pH ‡ | Polymorph(s) | Relative abundance§ | Refinement stage |
|---|---|---|---|---|---|---|---|
| 1 | Ac-1–140* | 2 | PBS† | 5.8 | 3B¶:3C | 93%:7% | 3B, see Tables 2 : 3C, 3D classification |
| 2 | Ac-1–140 | 6 | PBS | 5.8 | 3B:3C | 54%:46% | 3D classification |
| 3 | Ac-1–140 | 3 | PBS+50 mM NaCl | 5.8 | 3B:3C | 47%:53% | 3C, see Table 2 : 3B, 3D classification |
| 4 | Ac-1–140 | 4 | PBS+100 mM NaCl | 5.8 | 3B:3C | 52%:48% | 3D classification |
| Ac-1–140 (E46K) | PBS+50 mM NaCl | 5.8 | 3D | See Table 2 | |||
| 6 | Ac-1–140 | 7 | PBS | 6.5 | 2A:2B:3B:3C | 25%:18%:31%:26% | Filament subset selection followed by 3D refinement to 3.51 Å, 4.73 Å, 4.56 Å, 4.06 Å respectively |
| 7 | Ac-1–140 | 4 | 20 mM Tris, 140 mM NaCl | 7.0 | 1A | 3D refinement 3.65 Å | |
| 8 | Ac-1–140 | 4 | PBS | 7.0 | 5A | See Table 2 | |
| 9 | Ac-1–140 | 5 | PBS | 7.0 | 1M | See Table 2 | |
| 10 | Ac-1–140 | 5 | PBS | 7.0 | Non-twisted** | ||
| 11 | Ac-1–140 | 7 | PBS | 7.0 | 2A:2B:1M | 30%:20%:50%‡ ‡ | Filament subset selection followed by 3D refinement to 4.2 Å, 4.9 Å and 4.8 Å respectively |
| 12 | Ac-1–140 | 7 | PBS | 7.0 | 2A:2B:1M | 16%:9%:75%‡ ‡ | Filament subset selection followed by 3D refinement to 5.4 Å, 6.4 Å and 5.8 Å respectively†† |
| 13 | Ac-1–140 | 8 | PBS | 7.0 | 2A:2B | 78%:22% | Filament subset selection - see Table 2†† |
| 14 | Ac-1–140 | 8 | PBS | 7.0 | 1A | 3D refinement to 2.95 Å | |
| 15 | Ac-1–140 | 8 | PBS | 7.0 | 1A | 3D refinement to 3.94 Å | |
| 16 | Ac-1–140 | 8 | PBS | 7.0 | Non-twisted and clumped** | ||
| 17 | Ac-1–140 | 4 | PBS | 7.4 | 1A | 3D refinement to 3.73 Å | |
| 18 (F3) | Ac-1–140 | 5 | PBS +5% pH 5.8 seeds | 7.4 | 1A | 3D refinement to 3.50 Å | |
| 19 (F4) | Ac-1–140 | 7 | PBS +5% pH 7.4 seeds | 5.8 | 3B:3C | 52%:48% | 3D classification |
-
*
All constructs are full-length, N-terminally acetylated (Ac).
-
†
PBS is a 10 mM phosphate buffer solution with 137 mM NaCl and 2.7 mM KCl.
-
‡
The pH was adjusted after dissolving the PBS tablet (Sigma-Aldrich) by the addition of HCl.
-
§
In samples for which more than one polymorph could be identified by 3D classification or filament subset selection in RELION.
-
¶
Underlining indicates data that was used for 3D refinement of the deposited maps and for building the deposited coordinates.
-
**
This data could not be analyzed by helical reconstruction as only non-twisted fibrils were present.
-
††
Filament subset selection was run after 2D classification of the entire set of auto-picked particles. The identified filament classes were individually 2D classified and these classes used to create an initial model with relion_helix_inimodel2d which was used for automated 3D refinement. In the case of dataset 13 followed by CTF refinement and Bayesian polishing in RELION.
-
‡ ‡
Due to the small size of these datasets, the relative abundances are not likely to be precise.
Table 2
Cryo-electron microscopy (cryo-EM) structure determination statistics.
| Polymorph | 1M | 2A | 2B | 3B | 3C | 5A | 3D (E46K) |
|---|---|---|---|---|---|---|---|
| Data collection | |||||||
| Pixel size [Å] | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 |
| Defocus range [µm] | –0.8 to –2.5 | 0.8 to –2.5 | 0.8 to –2.5 | –0.8 to –2.5 | –0.8 to –2.5 | –0.8 to –2.5 | –0.8 to –2.5 |
| Voltage [kV] | 300 | 300 | 300 | 300 | 300 | 300 | 300 |
| Number of frames | 40 | 40 | 40 | 40 | 40 | 40 | 40 |
| Total dose [e-/Å2] | 69 | 58.5 | 58.5 | 62 | 67 | 75 | 65 |
| Reconstruction | |||||||
| Reconstruction box width [pixels] | 256 | 200 | 200 | 512 | 256 | 256 | 512 |
| Inter-box distance [Å] | 33 | 33 | 33 | 33 | 33 | 33 | 33 |
| Reconstruction pixel size [Å] | 1.3 | 1.3 | 1.3 | 0.65 | 1.3 | 1.3 | 0.65 |
| Micrographs | 1,624 | 1339 | 1339 | 7,729 | 3,127 | 1,850 | 4,666 |
| Initially extracted segments | 84,666 | 238,570 | 238,570 | 279,929 | 159,308 | 109,817 | 287,018 |
| Segments after 2D and 3D classification | 19,800 | 91,238 | 26,426 | 178,710 | 28,022 | 86,219 | 40,181 |
| 3D refinement resolution [Å] (FSC>0.143) | 3.58 | 2.99 | 3.13 | 2.64 | 3.41 | 3.40 | 2.40 |
| Final resolution [Å] (FSC >0.143) | 3.26 | 2.86 | 2.95 | 2.23 | 3.41 | 3.30 | 2.31 |
| Estimated map sharpening B-factor [Å2] | –43.8 | –73.0 | –74.9 | –51.7 | –101.7 | –87.4 | –32.8 |
| Axial symmetry | C1 | C2 | C1 | C1 | C2 | C1 | C1 |
| Helical rise [Å] | 4.79 | 4.82 | 2.39 | 2.37 | 4.77 | 2.42 | 2.41 |
| Helical twist [°] | –0.95 | –0.80 | 179.6 | 179.5 | –0.995 | 179.6 | 179.5 |
| Model composition and validation | |||||||
| Non-hydrogen atoms (5 layers) | 2515 | – | – | 4550 | 4500 | 6690 | 4450 |
| Protein residues (5 layers) | 365 | – | – | 640 | 650 | 960 | 630 |
| R.m.s. deviations bond length [Å] | 0.008 | – | – | 0.005 | 0.007 | 0.006 | 0.007 |
| R.m.s. deviations bond angles [°] | 1.166 | – | – | 0.637 | 0.976 | 0.863 | 1.093 |
| MolProbity score | 1.66 | – | – | 2.01 | 1.33 | 1.64 | 1.60 |
| Clashscore | 3.71 | – | – | 7.42 | 1.62 | 4.72 | 3.57 |
| Rotamer outliers [%] | 0 | – | – | 2.22 | 1.3 | 0 | 0.68 |
| Ramachandran plot favored [%] | 91.6 | – | – | 95.2 | 95.2 | 94.2 | 92.8 |
| Ramachandran plot allowed [%] | 81.4 | – | – | 4.8 | 4.8 | 5.8 | 7.2 |
| Ramachandran plot disallowed [%] | 0 | – | – | 0 | 0 | 0 | 0 |
| Model resolution [Å] (FSC >0.143/0.5) | 3.1/3.3 | – | – | 2.0/2.2 | 3.1/3.3 | 3.1/3.4 | 2.0/2.4 |
| PDB code | 8PK2 | – | – | 9FYP | 8PIX | 8PK4 | 8PJO |
| EMDB-ID | 17723 | 50860 | 50077 | 50888 | 17693 | 17726 | 17714 |
